Targeted enzymatic degradation of quorum-sensing peptides

ABSTRACT

Methods and compositions for the treatment of biofilms and/or the inhibition of biofilm formation. In one embodiment, a biofilm is treated and/or biofilm formation is inhibited by a method comprising contacting a biofilm or a surface with a bifunctional ligand comprising a quorum-sensing-peptide-binding region and a protease-binding region, whereby the biofilm is treated and/or biofilm formation on the surface is inhibited.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional PatentApplication No. 61/613,306 entitled, “TARGETED ENZYMATIC DEGRADATION OFQUORUM-SENSING PEPTIDES,” filed Mar. 20, 2012, the disclosure of whichis hereby incorporated herein by reference.

FIELD

The present invention relates generally to the fields of microbiologyand wound care. More particularly, it concerns methods and compositionsfor inhibiting biofilms in wounds and on medical devices.

BACKGROUND

Infections due to biofilm growth formation are common complications ofwound healing and surgical procedures, particularly in patients withdevitalized tissue and decreased immunity. A biofilm forms whenmicroorganisms, such as bacteria, fungi, yeast, protozoa, adhere to eachother and to a surface, and produce extracellular polymers thatfacilitate adhesion and provide a structural matrix. Biofilms may formon living tissue or inert, nonliving material.

Biofilm-associated microorganisms behave differently from planktonicorganisms. In particular, biofilms are characterized by their ability tobecome increasingly resistant to antimicrobial treatments. Accordingly,there is a need for methods and compositions that can be used in thetreatment of biofilms and/or in inhibiting the formation of biofilms.

SUMMARY

The present invention provides methods and compositions for thetreatment of biofilms and/or the inhibition of biofilm formation. In oneembodiment, the present invention provides a method for treating abiofilm and/or inhibiting biofilm formation comprising contacting abiofilm or a surface with a bifunctional ligand comprising aquorum-sensing-peptide-binding region and a protease-binding region,whereby the biofilm is treated and/or biofilm formation on the surfaceis inhibited. In various embodiments the biofilm may be located on asurface, such as a surgical instrument, infected hardware, or animplanted device, including an indwelling medical device such as acatheter or a ventilation tube, and the tissue may be in contact withthe device. In some embodiments, the patient is a human patient.

In certain embodiments, the method comprises contacting the biofilm orthe surface with two or more bifunctional ligands. The two or morebifunctional ligands may be applied simultaneously or consecutively. Themethod further comprises contacting the surface with a protease, anantibiotic, or a combination thereof. The surface may be a tissue siteis or includes a wound. The biofilm comprises Gram-positive bacteriaand/or Gram-negative bacteria and in particular embodiments, the biofilmcomprises Staphylococcus species such as S. epidermidis and S. aureus;Candida species such as Candida albicans; enterococci species such asEnterococcus faecalis, Streptococcus species; P. aeruginosa; K.pneumoniae; and diphtheroids. In a further embodiment, the methodfurther comprises applying reduced pressure to the tissue site.

In another embodiment, the present invention provides a bifunctionalligand comprising a quorum-sensing-molecule-binding region and aprotease-binding region. In a further embodiment, the present inventionprovides a composition comprising a bifunctional ligand and apharmaceutically acceptable carrier. In certain embodiment, thecomposition comprises two or more bifunctional ligands.

The biofilm may comprise one or more of a bacteria, fungi, yeast, orprotozoa. In one embodiment, the biofilm comprises Gram-positivebacteria. The Gram-positive bacteria may be, for example, one or more ofStaphylococcus aureus, Staphylococcus epidermis, Streptococcus pyogenes(group A), Streptococcus species (viridans group), Streptococcusagalactiae (group B), Streptococcus bovis, Streptococcus pneumoniae,Enterococcus species, Bacillus anthracis, Corynebacterium diphtheriae,Listeria monocytogenes, Clostridium tetani, or Clostridium difficile.The Gram-positive bacteria may be selected from the group consisting ofStaphylococcus aureus, Staphylococcus epidermis, Streptococcus pyogenes(group A), Streptococcus species (viridans group), Streptococcusagalactiae (group B), Streptococcus bovis, Streptococcus pneumoniae,Enterococcus species, Bacillus anthracis, Corynebacterium diphtheriae,Listeria monocytogenes, Clostridium tetani, and Clostridium difficile.

In some embodiments, the biofilm comprises Gram-negative bacteria. TheGram-negative bacteria may be, for example, one or more of Escherichiacoli, Enterobacter species, Proteus mirablis, Pseudomonas aeruginosa,Klebsiella pneumoniae, Salmonella species, Shigella species, Serratiaspecies, Campylobacterjejuni species, Neisseria species, or Branhamellacatarrhalis. The Gram-negative bacteria may be selected from the groupconsisting of Escherichia coil, Enterobacter species, Proteus mirablis,Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella species,Shigella species, Serratia species, Campylobacterjejuni species,Neisseria species, and Branhamella catarrhalis.

The surface being treated may be any surface on which a biofilm hasformed or may form. In certain aspects of the invention, the surface isliving tissue. In some embodiments, living tissue is in contact with anobject such as an indwelling medical device or a wound dressing.Non-limiting examples of indwelling medical devices include catheters,ventilation tubes, and feeding tubes. In some embodiments, the biofilmand/or the surface is located in a wound.

The quorum-sensing-molecule-binding region of the bifunctional ligand isa region that binds quorum-sensing molecules. The quorum sensingmolecule may be of bacterial origin, fungal origin, including yeastorigin, or protozoa origin. Non-limiting examples of quorum-sensingmolecules include acylated homoserine lactones (AHLs), autoinducingpeptides (AIP), AI-2 (a furanosyl borate diester), γ-butyrolactones, orcompetence stimulating peptide (CSP). Thequorum-sensing-molecule-binding region of the bifunctional ligand may bea small molecule, RNA- or DNA-based aptamer, or it may be derived fromthe naturally occurring transporter, receptor, or sensor of thequorum-sensing molecule. For example, LuxR is a sensor of AHL, ABCexporter and AgrC transmembrane sensor kinase bind AIP, Lsr ABC-typetransporter and LuxQ transmembrane sensor kinase bind AI-2, ArpA bindsγ-butyrolactone, and ComAB and ComD transmembrane sensor kinase bindCSP. Alternatively, the quorum-sensing-molecule-binding region of thebifunctional ligand may be a peptide or an antibody or antibody fragmentthat binds the quorum-sensing molecule.

The protease-binding region of the bifunctional ligand is a region thatbinds a protease capable of hydrolyzing a quorum-sensing peptide. Incertain aspects, the protease is derived from a microbe, such as abacteria, fungi, or protozoa. In certain embodiments, the protease is alactonase (also known as acyl-homoserine lactonase). Theprotease-binding region may be a peptide or an antibody or antibodyfragment that binds the quorum-sensing molecule.

The regions of the bifunctional ligand may be chemically joined. Incertain aspects, it may be desirable to include a linker or spacerbetween the regions in order to, for example, reduce steric hindrancebetween the two regions.

In some embodiments, the bifunctional ligands may be provided in avariety of forms, particularly forms suitable for topical delivery towound sites or application to medical devices that will be in contactwith tissues. For example, the bifunctional ligand may be applied bycoating the tissue with a liquid, gel, or foam formulation and injectedinto the tissue site. The bifunctional ligand may also be applied on oraround medical devices inserted in the body. The wound may be coveredwith a wound dressing after the bifunctional ligand is applied. Incertain aspects, the wound dressing comprises the bifunctional ligand(e.g., foam comprising the bifunctional ligand or gauze soaked in orcoated with the bifunctional ligand), in which case the bifunctionalligand may be applied to the wound by applying the wound dressing to thewound.

The composition may also include additional active ingredients, such asa protease or an additional antibiotic agent. The compositions disclosedherein may be provided in a kit.

It has been reported that sub-inhibitory concentrations of antimicrobialagents may induce biofilm formation (e.g., Frank et al., 2007). In viewof this, the lethal dosage for treatment of biofilm-formingmicroorganisms may be significantly higher than the standardtherapeutically effective amount determined for planktonicmicroorganisms (i.e., a lethal amount or a lethal dosage) typically usedby one of ordinary skill in the art. Thus, the “standard therapeuticallyeffective” amount would be the amount of antimicrobial agent necessaryto treat biofilm-forming microorganisms. A “standard therapeutic amount”or “standard therapeutic dose” may also refer to an amount of an agentsufficient to reduce or eliminate planktonic microorganisms. In someembodiments, treatment of biofilms and biofilm-forming microorganismsmay require two or more doses of the bifunctional ligand.

In some embodiments the bifunctional ligand is used in combination withother treatments. For example, in one embodiment, the method oftreatment comprises contacting the biofilm or the surface with thebifunctional ligand and a protease or an additional antibiotic agent.Where the area being treated is at a tissue site, the treatment mayfurther comprise applying reduced pressure to a wound.

The term “tissue site” as used herein includes, without limitation, awound or defect located on or within any tissue, including but notlimited to, bone tissue, adipose tissue, muscle tissue, neural tissue,dermal tissue, vascular tissue, connective tissue, cartilage, tendons,or ligaments. A wound may include chronic, acute, traumatic, subacute,and dehisced wounds, partial-thickness burns, ulcers (such as diabetic,pressure, or venous insufficiency ulcers), flaps, and grafts, forexample. The term “tissue site” may further refer to areas of any tissuethat are not necessarily wounded or defective, but are instead areas inwhich it is desired to add or promote the growth of additional tissue.For example, reduced pressure tissue treatment may be used in certaintissue areas to grow additional tissue that may be harvested andtransplanted to another tissue location. The tissue may be that of anymammal, such as a mouse, rat, rabbit, cat, dog, pig, or primate,including humans, that are being treated as patients. Also, the wound atthe tissue site may be due to a variety of causes, including trauma,surgery, degeneration, and other causes.

The term “medical indwelling device” refers to any medical deviceimplanted or inserted in the human body. Such devices can be temporarilyor permanently implanted or inserted.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The term “reduced pressure” as used herein generally refers to apressure less than the ambient pressure at a tissue site that is beingsubjected to treatment. In most cases, the reduced pressure will be lessthan the atmospheric pressure at which the patient is located.Alternatively, the reduced pressure may be less than a hydrostaticpressure associated with tissue at the tissue site. Although the terms“vacuum” and “negative pressure” may be used to describe the pressureapplied to the tissue site, the actual pressure reduction applied to thetissue site may be significantly less than the pressure reductionnormally associated with a complete vacuum. Reduced pressure mayinitially generate fluid flow in the area of the tissue site. As thehydrostatic pressure around the tissue site approaches the desiredreduced pressure, the flow may subside, and the reduced pressure is thenmaintained.

Instillation of a tissue site, which generally refers to the slowintroduction of a solution to the tissue site, can expose a tissue siteto temperature variations, drugs, or other substances that may furtherpromote healing or growth of tissue. Instillation may also be referredto as irrigation or infusion in some contexts. Instillation may becontinuous or intermittent and may take place prior to, subsequent to,or simultaneously with the application of reduced pressure. In someembodiments, instillation and negative pressure may be coordinated by acentral controller.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, amethod, composition, kit, or system that “comprises,” “has,” “contains,”or “includes” one or more recited steps or elements possesses thoserecited steps or elements, but is not limited to possessing only thosesteps or elements; it may possess (i.e., cover) elements or steps thatare not recited. Likewise, an element of a method, composition, kit, orsystem that “comprises,” “has,” “contains,” or “includes” one or morerecited features possesses those features, but is not limited topossessing only those features; it may possess features that are notrecited.

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Targeted enzymatic degradation of quorum-sensing peptides usinga bifunctional ligand.

FIG. 2 shows a block diagram of a representative embodiment of a therapysystem.

DETAILED DESCRIPTION

A biofilm forms when microorganisms adhere to each other and to asurface, and produce extracellular polymers that facilitate adhesion andprovide a structural matrix. This surface may be living tissue or inert,nonliving material. Formation of the biofilm begins with the initialattachment of microorganisms to a surface. The attachment is initiallythrough weak, reversible adhesion, but the microorganisms will proceedto anchor themselves more permanently using cell adhesion structuressuch as pili if they are not removed from the surface. Other cells thenarrive to build the matrix that holds the biofilm together. Some speciesare not able to attach to a surface on their own, but are able to anchorthemselves to the matrix or directly to earlier colonists. Thus, theearly colonists provide adhesion sites that facilitate the adhesion ofadditional cells.

During colonization, the cells communicate via quorum sensing, which isa system of stimulus and response correlated to population density. Avariety of different molecules can be used as signals in quorum sensing.These signaling molecules are also referred to as “quorum-sensingmolecules,” “inducer molecules,” or “autoinducers.” Microbes also havereceptors that “sense” the presence or absence of quorum-sensingmolecules. Common classes of quorum-sensing molecules include N-AcylHomoserine Lactones (AHL) in Gram-negative bacteria, and a family ofautoinducers known as autoinducer-2 (AI-2) in both Gram-negative andGram-positive bacteria.

P. aeruginosa, for example, relies on quorum sensing via production ofsignaling molecules such as lactones N-butanoyl-1-homoserine (C4-HSL)and N-(3-oxododecanoyl)-1-HSL (3-oxo-C12-HSL) (Smith et al. (2003)Current Opinion in Microbiology 1(1): 56-60). Although multiplesignaling molecules may be involved in quorum sensing, the absence ofjust one component of the quorum-sensing system can result in asignificant reduction in biofilm formation.

As mentioned above, the microorganisms also have receptors for thesignaling molecules. For example, the receptor LuxP is a receptor forAI-2. When the signaling molecule binds the receptor, it activatestranscription of certain genes, including those for signaling moleculesynthesis forming a positive feedback loop.

Quorum quenching may be achieved by degrading the signaling molecule,such as with a protease. Lactonase (also known as acyl-homoserinelactonase) is a metalloenzyme that targets and inactivates acylatedhomoserine lactones (AHLs). Lactonase hydrolyzes the ester bond of thehomoserine lactone ring of acylated homoserine lactones. By hydrolysingAHL, lactonase prevents these signaling molecules from binding to theirtarget transcriptional regulators, thereby inhibiting quorum sensing.Lactonases have been reported for Bacillus, Agrobacterium, Rhodococcus,Streptomyces, Arthrobacter, Pseudomonas, and Klebsiella (Schipper et al.(2009) Molecular Plant-Microbe Interactions 75: 224-233). The Bacilluscereus group (B. cereus, B. thuringiensis, B. mycoides, and B.anthracis) was found to contain nine genes homologous to the AiiA genethat encode AHL-inactivating enzymes (Dong et al. (2002). Applied andEnvironmental Microbiology 68(4): 1754-1759). Some examples oflactonases are AiiA produced by Bacillus species, AttM and AiiB producedby Agrobacterium tumefaciens, and QIcA produced by Rhizobiales species(Riaz et al. (2008) Environmental Microbiology 10(3): 560-570). Thedirect therapeutic application of lactonases is complicated by the factthat lactonolysis is a reversible reaction (Rasmussen and Givskov (2006)International Journal of Medical Microbiology 296(2-3):149-161). Thebifunctional ligand disclosed herein provides a solution to this problemby targeting proteases, such as lactonases, to the quorum sensingsignaling molecules and thereby mediating irreversible degradation ofthe molecule.

Biofilm-associated microorganisms behave differently from planktonicorganisms. Additionally, biofilms are characterized by their ability tobecome increasingly resistant to antimicrobial treatments. Resistance ofbiofilms to antimicrobial agents is believed to be due to theextracellular matrix in which the bacterial cells are embedded providinga barrier toward penetration by the biocides (Costerton et al., 1999).However, it is also possible that a majority of the cells in a biofilmare in a slow-growing, nutrient-starved state, and therefore not assusceptible to the effects of anti-microbial agents. Additionally, theresistance to antimicrobial agents may be due to the cells in a biofilmadopting a distinct and protected biofilm phenotype, e.g., by elevatedexpression of drug-efflux pumps. Antimicrobial concentrations sufficientto inactivate planktonic organisms are generally inadequate toinactivate biofilm organisms, especially those deep within the biofilm.Biofilm formations also make it difficult for host phagocytic cells togain access to and kill the organisms.

Biofilms can be comprised of bacteria, fungi, yeast, protozoa, and othermicroorganisms. Biofilms may be composed of single species or multiplespecies of microorganism. The most common biofilms have been found to bebacterial biofilms. Both Gram-negative and Gram-positive bacteria arecapable of forming biofilms. Examples of Gram-positive bacteria that arecapable of forming biofilms include, but are not limited to,Staphylococcus aureus, coagulase negative staphylocci such asStaphylococcus epidermis, Streptococcus pyogenes (group A),Streptococcus species (viridans group), Streptococcus agalactiae (groupB), S. bovis, Streptococcus (anaerobic species), Streptococcuspneumoniae, and Enterococcus species. Other Gram-positive bacillicapable of forming biofilms include Bacillus anthracis, Corynebacteriumdiphtheriae and Corynebacterium species which are diptheroids (aerobicand anerobic), Listeria monocytogenes, Clostridium tetani, andClostridium difficile. Examples of Gram-negative bacteria that arecapable of forming biofilms are bacteria from the genus Escherichiacoli, Enterobacter species, Proteus mirablis and other species,Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Shigella,Serratia, and Campylobacterjejuni, Neisseria and Branhamellacatarrhalis.

Additional organisms capable of forming biofilm may includedermatophytes (Microsporum canis and other M. spp.; and Trichophytonspp. such as T. rubrum, and T. mentagrophytes), yeasts (e.g., Candidaalbicans, C. Parapsilosis, C. glabrata, C. tropicalis, or other Candidaspecies including drug resistant Candida species),Epidermophytonfloccosum, Malassezia fuurfur (Pityropsporon orbiculare,or P. ovale), Cryptococcus neoformans, Aspergillus fumigatus, and otherAspergillus spp., Zygomycetes (Rhizopus, Mucor), hyalohyphomycosis(Fusarium Spp.), Paracoccidioides brasiliensis, Blastomycesdermatitides, Histoplasma capsulatum, Coccidioides immitis, andSporothrix schenckii.

Infections due to biofilm growth formation are common complications ofwound healing and surgical procedures, particularly in patients withdevitalized tissue and decreased immunity. The organisms causing biofilmgrowth formation that are most commonly isolated from indwelling medicaldevices include Staphylococcus species such as S. epidermidis and S.aureus; Candida species such as Candida albicans; enterococci speciessuch as Enterococcus faecalis, Streptococcus species; P. aeruginosa; K.pneumoniae; and diphtheroids. These organisms may originate from theskin of patients or healthcare workers, tap water to which entry portsare exposed, or other sources in the environment.

The predilection of Pseudomonas aeruginosa to form biofilms is a majorcontributing factor to the problems of biofilm growth formation in themedical and industrial environments. P. aeruginosa is highly associatedwith biofilm growth and catheter obstruction. For example, biofilms ofP. aeruginosa have been isolated from medical implants, such asindwelling urethral, venous or peritoneal catheters (Stickler et al.,1998). Burn wounds often become infected with P. aeruginosa from whichlife-threatening invasion of the blood stream and septic shock canarise. These infections are believed to involve the formation ofbiofilms. Thus, topical application to the wounds of a bifunctionalligand composition as described herein can be used in to inhibitinitiation of infection by preventing or inhibiting biofilm formation.

As discussed above, the bifunctional ligands disclosed herein comprise afirst region that binds to a quorum-sensing molecule and a second regionthat binds a protease. The quorum-sensing-molecule-binding region of thebifunctional ligand may be a small molecule or derived from, forexample, the naturally occurring receptor of the quorum-sensing moleculeor an antibody or antibody fragment that binds the quorum-sensingmolecule. The protease-binding region binds a protease capable ofhydrolyzing the quorum-sensing molecule that binds to thequorum-sensing-molecule-binding region of the bifunctional ligand.

Quorum-sensing molecules include, for example, acylated homoserinelactones (AHLs), autoinducing peptides (AIP), AI-2 (a furanosyl boratediester), γ-butyrolactones, or competence stimulating peptide (CSP).Various microbes may use the above-mentioned quorum-sensing systems.Non-limiting examples of bacteria that use each of the above-mentionedsystems are: Vibrio fischeri (AHL), Staphylococcus aureus (AIP), Vibrioharveyi (AI-2), Streptomyces griseus (γ-butyrolactone), andStreptococcus pneumonia (CSP). The quorum-sensing-molecule-bindingregion of the bifunctional ligand may be a small molecule or it may bederived from the naturally occurring transporter, receptor, or sensor ofthe quorum-sensing molecule. For example, LuxR is a sensor of AHL, ABCexporter and AgrC transmembrane sensor kinase bind AIP, Lsr ABC-typetransporter and LuxQ transmembrane sensor kinase bind AI-2, ArpA bindsγ-butyrolactone, and ComAB and ComD transmembrane sensor kinase bindCSP. Alternatively, the quorum-sensing-molecule-binding region of thebifunctional ligand may be a peptide or an antibody or antibody fragmentthat binds the quorum-sensing molecule.

The protease-binding region may, for example, bind a lactonase.Lactonases are metalloenzymes that target and inactivate acylatedhomoserine lactones (AHLs). Accordingly, a bifunctional ligand maycomprise a quorum-sensing-molecule-binding region that binds an AHL anda protease-binding region that binds a lactonase that hydrolyzes theAHL. Such a bifunctional ligand would, therefore, promote theinteraction of lactonase and AHL. By hydrolysing AHL, lactonase preventsthese signaling molecules from binding to their target transcriptionalregulators, thereby inhibiting quorum sensing. Lactonases have beenreported for Bacillus, Agrobacterium, Rhodococcus, Streptomyces,Arthrobacter, Pseudomonas, and Klebsiella (Schipper et al. (2009)Molecular Plant-Microbe Interactions 75: 224-233). The Bacillus cereusgroup (B. cereus, B. thuringiensis, B. mycoides, and B. anthracis) wasfound to contain nine genes homologous to the AiiA gene that encodeAHL-inactivating enzymes (Dong et al. (2002) Applied and EnvironmentalMicrobiology 68 (4): 1754-1759). Some examples of lactonases are AiiAproduced by Bacillus species, AttM and AiiB produced by Agrobacteriumtumefaciens, and QIcA produced by Rhizobiales species (Riaz et al.(2008) Environmental Microbiology 10 (3): 560-570).

The bifunctional ligand may further comprise a linker region between thequorum-sensing-molecule-binding region and the protease-binding region.The linker can be used to modify the distance and flexibility betweenthe quorum-sensing-molecule-binding region and the protease-bindingregion. The linker may be, for example, a peptide of about 1 to about 50amino acids in length.

The bifunctional ligands may be produced by techniques know to those inthe art. For example, the bifunctional ligands may be prepared bychemical synthesis, recombinant DNA techniques, or by chemical linkageof the domains.

As discussed above, the quorum-sensing-molecule-binding region of thebifunctional ligand may comprise a small molecule. A small molecule is alow molecular weight (less than approximately 800 Daltons) organiccompound. Small molecules are not polymers, but they may bind topolymers such as proteins. High-through put screening of small moleculelibraries may be performed to identify small molecules that bind aparticular quorum-sensing molecule. A wide variety of small moleculelibraries are available from commercial vendors as well as through theNational Institutes of Health. Alternatively, one may create a smallmolecule library using, for example, combinatorial synthesis methods. Avariety of assay formats are known for screening small moleculelibraries, and these assays may be employed for identifying smallmolecules that bind quorum-sensing molecules.

One approach for identifying a suitable protease-binding region is phagedisplay. Phage display is a well-known technique that usesbacteriophages for the high-throughput study of protein-proteininteractions. Using phage display, large libraries of proteins can bescreened for their ability to bind a particular protease.

The bifunctional ligands may be provided in a variety of forms,particularly forms suitable for topical delivery to wound sites orapplication to medical devices that will be in contact with tissues. Forexample, the bifunctional ligand may be formulated in a pharmaceuticalcomposition. The bifunctional ligand may be formulated into all types ofvehicles. Non-limiting examples of suitable vehicles include emulsions(e.g., oil-in-water, water-in-oil, silicone-in-water, water-in-silicone,water-in-oil-in-water, oil-in-water, oil-in-water-in-oil,oil-in-water-in-silicone, etc.), creams, lotions, solutions (bothaqueous and hydro-alcoholic), anhydrous bases (such as lipsticks andpowders), gels, ointments, pastes, milks, liquids, aerosols, solidforms, sprays, hydrogels, or electroporation device cartridges. In someembodiments, the formulation may be a hydrophilic solution, athixotropic spray, or other hydrophillic topical. Variations and otherappropriate vehicles will be apparent to the skilled artisan and areappropriate for use in the present invention. In certain aspects, theconcentrations and combinations of the ingredients can be selected insuch a way that the combinations are chemically compatible and do notform complexes which precipitate from the finished product. In stillother aspects, the formulation may be immobilized on a surface, such asa dressing, and activated by a glucose wash.

The composition may be applied through infusion within, injection into,absorption by, layering on, encapsulation within, or coating on, acarrier material, such as a bandage, gauze, wound dressing, adhesivebandage, scaffold, or hydrogel, for instance, comprising cellulosederivatives, including hydroxyethyl cellulose, hydroxymethyl cellulose,carboxymethyl cellulose, hydroxypropylmethyl cellulose and mixturesthereof; and hydrogels containing polyacrylic acid (Carbopols) as wellas gelatin. For example, a bifunctional ligand composition may beapplied to a woven, non-woven, or knitted fabric material, such asgauze, dispersed within film, sponge, or foam for sustained release at atissue site. A “carrier material” as used herein refers to a materialsuitable for a bifunctional ligand composition. The carrier material maybe either bioresorbable, for instance comprising polyglycolic acid,polylactic acid, polydioxanone, polyhydroxybutyrate,polyhydrozyvalerate, polyaminoacids polyorthoesters, polyvinly alcohol,collagen, gelatin, chitosan, oxidized regenerated cellulose, hyaluronicacid, alginate or derivatives thereof, or may be non-bioresorbable,comprising for instance, polyurethane, polyvinyl alcohol, or gauze. Incertain aspects, the composition may be bound to the carrier material,such as through hydrogen binding, covalent binding or ionic binding.

Additionally, the bifunctional ligand may be formulated in a compositionwith one or more additional active ingredients. For example, thebifunctional ligand may be formulated in a composition with one or moreantibiotics. Such a formulation will be advantageous in treating orpreventing infection because the bifunctional ligand will inhibitbiofilm formation thus allowing the antibiotic to more effectively killthe microbes. Additionally, two or more different bifunctional ligandsmay be combined in a composition. The composition may also be aninstillation composition. The compositions may be delivered to a tissuesite by continuous instillation and/or periodic instillation. Theinstillant provides fresh bifunctional ligand to a tissue site such as awound.

The method for applying the bifunctional ligand to tissue site or woundmay vary depending on factors such as the location and the size andshape of the area to be treated. A health care provider will be able todetermine an appropriate method for applying the bifunctional ligand tothe area in view of such factors.

The treatment methods of the present invention may be used on their ownor in combination with additional methods of treatment. In order toincrease the effectiveness of a treatment with the compositions andmethods of treatment of the present invention or to augment theprotection of another (second) therapy, it may be desirable to combinethese compositions and methods with other agents and methods effectivein the treatment, reduction of risk, or prevention of infections, forexample, anti-bacterial, anti-viral, and/or anti-fungal treatments. Asanother example, iontophoresis can be used to drive agents into tissuesfor the purpose of labeling or eradicating biofilms.

Yet another example would be the use of the treatment integrated withnegative pressure wound therapy (NPWT), such as V.A.C.® Therapy (KCIInternational, San Antonio, Tex.), fluid instillation therapy, or both.Clinical studies and practice have shown that NPWT can augment andaccelerate growth of new tissue at a tissue site. The applications ofthis phenomenon are numerous, but it has proven particularlyadvantageous for treating wounds. Regardless of the etiology of a wound,whether trauma, surgery, or another cause, proper care of the wound isimportant to the outcome. Treatment of wounds with reduced pressure maybe commonly referred to as NPWT, but is also known by other names,including “negative-pressure therapy,” “reduced-pressure wound therapy,”“vacuum therapy,” and “vacuum-assisted closure,” for example.Negative-pressure therapy may provide a number of benefits, includingmigration of epithelial and subcutaneous tissues, improved blood flow,and micro-deformation of tissue at a tissue site. Together, thesebenefits can increase development of granulation tissue and reducehealing times.

Instillation of a tissue site, which generally refers to the slowintroduction of a solution to the tissue site, can expose a tissue siteto temperature variations, drugs, or other substances that may furtherpromote healing or growth of tissue. Instillation may also be referredto as irrigation or infusion in some contexts. Instillation may becontinuous or intermittent and may take place prior to, subsequent to,or simultaneously with the application of negative pressure. In someembodiments, instillation and negative pressure may be coordinated by acentral controller.

FIG. 2 is a simplified functional block diagram of an example embodimentof a therapy system 100 that can provide therapeutic pressure andinstillation in accordance with this specification. As illustrated, thetherapy system 100 may include a dressing 102 fluidly coupled to anegative-pressure source 104. A regulator or controller, such asregulator 106, may also be fluidly coupled to the dressing 102 and thenegative-pressure source 104. The dressing 102 generally includes adrape, such as drape 108, and a manifold, such as distribution manifold110. The therapy system 100 may also include fluid containers, such ascontainer 112 and container 114, coupled to the dressing 102. Asillustrated in FIG. 2, container 112 may be also be fluidly coupled tothe negative-pressure source 104 in some embodiments, and container 114may be coupled to a fluid-delivery device, such as a pump 116.

In general, components of the therapy system 100 may be coupled directlyor indirectly. For example, the negative-pressure source 104 may bedirectly coupled to regulator 106 and indirectly coupled to dressing 102through regulator 106. Components may be fluidly coupled to each otherto provide a path for transferring fluids (i.e., liquid and/or gas)between the components. In some embodiments, components may be fluidlycoupled with a tube, for example. A “tube,” as used herein, broadlyrefers to a tube, pipe, hose, conduit, or other structure with one ormore lumina adapted to convey fluids between two ends. Typically, a tubeis an elongated, cylindrical structure with some flexibility, but thegeometry and rigidity may vary. In some embodiments, components mayadditionally or alternatively be coupled by virtue of physicalproximity, being integral to a single structure, or being formed fromthe same piece of material. Coupling may also include mechanical,thermal, electrical, or chemical coupling (such as a chemical bond) insome contexts.

In operation, the distribution manifold 110 may be placed within, over,on, or otherwise proximate to a tissue site. The drape 108 may be placedover the distribution manifold 110 and sealed to tissue proximate to thetissue site. The tissue proximate to the tissue site is often undamagedepidermis peripheral to the tissue site. Thus, the dressing 102 canprovide a sealed therapeutic environment proximate to a tissue site,substantially isolated from the external, ambient environment. Thenegative-pressure source 104 can reduce the pressure in the sealedtherapeutic environment, and the pump 116 can apply therapeuticsolutions, including the embodiments of the bifunctional ligandcompositions described herein. Reduced pressure and/or fluids can beapplied substantially uniformly through the distribution manifold 110 inthe sealed therapeutic environment. Reduced pressure can inducemacrostrain and microstrain in the tissue site, as well as removeexudates and other fluids from the tissue site, which can be collectedin the container 112 and disposed of properly.

Integrating negative pressure therapy and instillation therapy withembodiments of bifunctional ligand compositions described herein canfurther promote healing and growth of tissue by removing barriers tonormal healing, such as the presence of biofilm and biofilm-formingbacteria. Functionally coupling infusion of the composition with reducedpressure therapy as disclosed herein provides unexpected decreases inwound bioburden and wound healing trajectories. The ability of agelatin-based composition to operate with a reduced pressure therapysystem allows for the use of the composition as an instillate to treat atissue site, in particular a chronic wound, with a composition thattreats and/or inhibits biofilm formation.

In some embodiments, the negative pressure with the bifunctional ligandcomposition can be applied during debridement of a tissue site, forexample, by using a dressing having a self-contained debriding mechanismsuch as ultrasound or pulsed lavage. Alternatively, negative pressuretherapy may be applied after debridement, to promote vascularstimulation and the formation of granulation tissue. Further still, thetransition from debridement to negative pressure therapy is seamless, aswell as from negative pressure therapy to passive infusion with thecomposition, that is, without disrupting the integrity of the tissuesite.

The negative pressure with the bifunctional ligand composition may alsobe applied during cleansing or irrigation of the wound in someembodiments. Alternatively, the negative pressure may be applied priorto or after the cleansing of the wound with the composition.

The compositions provided herein can be used in conjunction with allcurrent NPWT devices, and delivered in either the inpatient oroutpatient setting. Exemplary negative pressure devices include V.A.C.®Therapy, V.A.C.® Instill, or V.A.C.® Ulta therapy systems (KineticConcepts, Inc.). These devices, or devices having similar or equivalentdesigns may be used.

As noted previously, “reduced pressure” or “negative pressure” generallyrefer to a pressure less than a local ambient pressure, such as theambient pressure in a local environment external to a sealed therapeuticenvironment provided by the dressing 102. In many cases, the localambient pressure may also be the atmospheric pressure in the vicinity ofa tissue site. Alternatively, the pressure may be less than ahydrostatic pressure associated with tissue at the tissue site. Unlessotherwise indicated, values of pressure stated herein are gaugepressures. Similarly, references to increases in reduced pressuretypically refer to a decrease in absolute pressure, while decreases inreduced pressure typically refer to an increase in absolute pressure.

A reduced-pressure source, such as the reduced-pressure source 104, maybe a reservoir of air maintained at a reduced pressure, or may be amanual or electrically-powered device that can reduce the pressure in asealed volume, such as a vacuum pump, a suction pump, a wall suctionport available at many healthcare facilities, or a micro-pump, forexample. A reduced-pressure source may be housed within or used inconjunction with other components, such as sensors, processing units,alarm indicators, memory, databases, software, display devices, or userinterfaces that further facilitate reduced-pressure therapy. While theamount and nature of reduced pressure applied to a tissue site may varyaccording to therapeutic requirements, the pressure typically rangesbetween −5 mm Hg (−667 Pa) and −500 mm Hg (−66.7 kPa). Commontherapeutic ranges are between −75 mm Hg (−9.9 kPa) and −300 mm Hg(−39.9 kPa).

A fluid-delivery device, such as the pump 116, may be a rotary-deliverypump, or other pump that can supply an instillation solution to a sealedspace or the distribution manifold 110. A fluid-delivery device may behoused within a therapy device or used in conjunction with othercomponents, such as sensors, processing units, alarm indicators, memory,databases, software, display devices, or user interfaces that furtherfacilitate instillation therapy. In some embodiments, a fluid-deliverydevice and a negative-pressure source may be integrated into a singleunit to provide both negative pressure and instillation, or toalternatingly supply negative pressure and instillation.

A manifold, such as the distribution manifold 110, can generally beadapted to contact a tissue site. The distribution manifold 110 may beadapted to be placed partially or fully in contact with the tissue site.If the tissue site is a wound, for example, the distribution manifold110 may partially or completely fill the wound, or may be placed overthe wound. The distribution manifold 110 may take many forms, and may bemany sizes, shapes, or thicknesses depending on a variety of factors,such as the type of treatment being implemented or the nature and sizeof a tissue site. For example, the size and shape of the distributionmanifold 110 may be adapted to the contours of deep and irregular shapedtissue sites.

More generally, a manifold is a substance or structure adapted todistribute negative pressure to or remove fluids from a tissue site, orboth. In some embodiments, though, a manifold may also facilitatedelivering fluids to a tissue site, if the fluid path is reversed or asecondary fluid path is provided, for example when instillation solutionis applied. A manifold may include flow channels or pathways thatdistribute fluids provided to and removed from a tissue site around themanifold. In one illustrative embodiment, the flow channels or pathwaysmay be interconnected to improve distribution of fluids provided to orremoved from a tissue site. For example, cellular foam, open-cell foam,porous tissue collections, and other porous material such as gauze orfelted mat generally include structural elements arranged to form flowchannels. Liquids, gels, and other foams may also include or be cured toinclude flow channels.

In one illustrative embodiment, the distribution manifold 110 may be aporous foam material having interconnected cells or pores adapted touniformly (or quasi-uniformly) distribute reduced pressure to a tissuesite. The foam material may be either hydrophobic or hydrophilic. In onenon-limiting example, the distribution manifold 110 may be an open-cell,reticulated polyurethane foam such as GranuFoam® dressing available fromKinetic Concepts, Inc. of San Antonio, Tex.

In some embodiments, such as embodiments in which the distributionmanifold 110 may be made from a hydrophilic material, the distributionmanifold 110 may also wick fluid away from a tissue site whilecontinuing to distribute reduced pressure to the tissue site. Thewicking properties of the distribution manifold 110 may draw fluid awayfrom a tissue site by capillary flow or other wicking mechanisms. Anexample of a hydrophilic foam is a polyvinyl alcohol, open-cell foamsuch as V.A.C. WhiteFoam® dressing available from Kinetic Concepts, Inc.of San Antonio, Tex. Other hydrophilic foams may include those made frompolyether. Other foams that may exhibit hydrophilic characteristicsinclude hydrophobic foams that have been treated or coated to providehydrophilicity.

The distribution manifold 110 may further promote granulation at atissue site if pressure within a sealed therapeutic environment isreduced. For example, any or all of the surfaces of the distributionmanifold 110 may have an uneven, coarse, or jagged profile that caninduce microstrains and stresses at a tissue site if reduced pressure isapplied through the distribution manifold 110.

In one example embodiment, the distribution manifold 110 may beconstructed from bioresorbable materials. Suitable bioresorbablematerials may include, without limitation, a polymeric blend ofpolylactic acid (PLA) and polyglycolic acid (PGA). The polymeric blendmay also include without limitation polycarbonates, polyfumarates, andcapralactones.

Other bioresorbable materials that may be used include, but are notlimited to, polydioxanone, polyhydroxybutyrate, polyhydrozyvalerate,polyaminoacids polyorthoesters, polyvinly alcohol, chitosan, oxidizedregenerated cellulose, hyaluronic acid, alginate, collagen, a modifiedcollagen, such as gelatin or derivatives of any of the above.

The distribution manifold 110 may further serve as a scaffold for newcell-growth, or a scaffold material may be used in conjunction with thedistribution manifold 110 to promote cell-growth. In general, a scaffoldmaterial may be a substance or structure used to enhance or promote thegrowth of cells or formation of tissue, such as a three-dimensionalporous structure that provides a template for cell growth.

A scaffold and/or manifold may be also be infused with, coated with, orcomprised of cells, growth factors, extracellular matrix components,nutrients, integrins, or other substances to promote cell growth inaddition to embodiments of the compositions described herein. Themanifold or scaffold may serve as a carrier material for the compositiondescribed herein.

Scaffolds may be formed from biologic or synthetic scaffold materials,and are used in the field of tissue engineering to support proteinadhesion and cellular ingrowth for tissue repair and regeneration. Thecurrent state of the art in scaffold technology relies upon the inherentcharacteristics of the surrounding tissue space for the adsorption ofproteins and migration of cells. Nonlimiting examples of suitablescaffold materials include extracellular matrix proteins such as fibrin,collagen or fibronectin, and synthetic or naturally occurring polymers,including bioabsorbable or non-absorbable polymers, such as polylacticacid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA),polyvinylpyrrolidone, polycaprolactone, polycarbonates, polyfumarates,caprolactones, polyamides, polysaccharides (including alginates (e.g.,calcium alginate) and chitosan), hyaluronic acid, polyhydroxybutyrate,polyhydroxyvalerate, polydioxanone, polyorthoesthers, polyethyleneglycols, poloxamers, polyphosphazenes, polyanhydrides, polyamino acids,polyacetals, polycyanoacrylates, polyurethanes (e.g., GranuFoam®),polyacrylates, ethylene-vinyl acetate polymers and other acylsubstituted cellulose acetates and derivatives thereof, polystyrenes,polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole),chlorosulphonated polyolefins, polyethylene oxide, polyvinyl alcohol,Teflon®, and nylon.

The scaffold can also comprise ceramics such as hydroxyapatite, corallinapatite, calcium phosphate, calcium sulfate, calcium carbonate or othercarbonates, bioglass, allografts, autografts, xenografts, decellularizedtissues, or composites of any of the above. In some embodiments, thescaffold may comprise collagen (e.g., Biostep™ or Pomogran™ scaffolds),polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide(PLGA), a polyurethane, a polysaccharide, an hydroxyapatite, or apolytherylene glycol. Additionally, the scaffold can comprisecombinations of any two, three or more materials, either in separate ormultiple areas of the scaffold, combined noncovalently or covalently(e.g., copolymers such as a polyethylene oxide-polypropylene glycolblock copolymers, or terpolymers), or combinations thereof.

The drape 108 is an example of a sealing member. A sealing member may beconstructed from a material that can provide a fluid seal between twoenvironments or components, such as between a therapeutic environmentand a local external environment. The sealing member may be, forexample, an impermeable or semi-permeable, elastomeric material that canprovide a seal adequate to maintain a negative pressure at a tissue sitefor a given negative-pressure source. For semi-permeable materials, thepermeability generally should be low enough that a desired negativepressure may be maintained. An attachment device may be used to attach asealing member to an attachment surface, such as undamaged epidermis, agasket, or another sealing member. The attachment device may take manyforms. For example, an attachment device may be a medically-acceptable,pressure-sensitive adhesive that extends about a periphery, a portion,or an entire sealing member. Other example embodiments of an attachmentdevice may include a double-sided tape, paste, hydrocolloid, hydrogel,silicone gel, organogel, or an acrylic adhesive.

The container 112 is representative of a container, canister, pouch, orother storage component, which can be used to manage exudates and otherfluids withdrawn from a tissue site. In many environments, a rigidcontainer may be preferred or required for collecting, storing, anddisposing of fluids. In other environments, fluids may be properlydisposed of without rigid container storage, and a re-usable containercould reduce waste and costs associated with negative-pressure therapy.

The container 114 is representative of another container, canister,pouch, cartridge, or other storage component, which can be used tomanage instillation solution to be supplied to a tissue site. In manyenvironments a rigid container may be preferred or required fordelivering, storing, and supplying of the instillation solution. Inother environments, instillation solution may be provided in a non-rigidcontainer, and a re-usable container could reduce waste and costsassociated with instillation.

Components of therapy system 100 may also be provides as one or morekits. In one embodiment, for example, a kit comprises components forinhibiting biofilms. Any of the components disclosed here in may becombined in a kit. In certain embodiments, the kit comprises abiofilm-inhibiting composition disclosed herein. The kit may furthercomprise a wound dressing, such as a gauze, cloth, or film.

The kits will generally include at least one vial, test tube, flask,bottle, syringe, foil package, or other container, into which acomponent may be placed, and preferably, suitably aliquoted. Where thereis more than one component in the kit, the kit also will generallycontain a second, third or other additional containers into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a container. The kits ofthe present invention also will typically include packaging forcontaining the various containers in close confinement for commercialsale. Such packaging may include cardboard or injection or blow moldedplastic packaging into which the desired containers are retained. A kitmay also include instructions for employing the kit components.Instructions may include variations that can be implemented.

The effectiveness of the treating a biofilm or inhibitingbiofilm-formation may be determined using methods known in the art. Onemethod is to extract any bacteria remaining in the biofilm aftertreatment, plating the bacteria on a suitable media and allowing thebacteria to grow for 24-48 hours or as appropriate, then counting anybacteria. A reduction in bacteria is reflects effectiveness of thebifunctional ligand and the method of treatment. Alternatively,treatment between groups using in vitro methods such as described inPhillips et al., “Effects of antimicrobial Agents on an in vitro biofilmmodel of skin wounds,” Advances in Wound Care, 1:299-304 (2010), or asotherwise known in the art.

EXAMPLES Example I

A subject is identified as having a wound that has become infected withS. aureus that has formed a biofilm in the wound. The wound is treatedwith a bifunctional ligand that comprises a first region that binds to aquorum-sensing molecule and a second region that binds a protease with astandard therapeutically effective amount for a period of timesufficient to reduce biofilm in the wound.

Example II

A subject is identified as having a wound that has become infected witha P. aeruginosa that has formed a biofilm in the wound. The wound istreated with a bifunctional ligand that comprises a first region thatbinds to a quorum-sensing molecule and a second region that binds aprotease with a standard therapeutically effective amount for a periodof time in combination with VAC® Therapy at −125 mmHg to reduce biofilmin the wound.

Example III

P. aeruginosa forms a biofilm on a ventilation tube. The ventilationtube is treated with a bifunctional ligand that comprises a first regionthat binds to a quorum-sensing molecule and a second region that binds aprotease in an amount and for a period of time sufficient to reduce thebiofilm on the ventilation tube.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of certain embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The invention claimed is:
 1. A method for treating a biofilm and/orinhibiting biofilm formation, comprising: contacting a biofilm or asurface with a bifunctional ligand comprising aquorum-sensing-molecule-binding region chemically linked to aprotease-binding region; and delivering reduced pressure to the biofilmor the surface; wherein the quorum-sensing-molecule-binding region isadapted to bind one or more of acylated homoserine lactones (AHLs),autoinducing pepties (AIP), AI-2 (furanosyl borate diester),γ-butyrolactones, or competence stimulating peptide (CSP); wherein theprotease-binding region is adapted from a lactonase.
 2. The method ofclaim 1, wherein the biofilm comprises Gram-positive bacteria.
 3. Themethod of claim 2, wherein the Gram-positive bacteria is one or more ofStaphylococcus aureus, Staphylococcus epidermis, Streptococcus pyogenes(group A), Streptococcus species (viridans group), Streptococcusagalactiae (group B), Streptococcus bovis, Streptococcus pneumoniae,Enterococcus species, Bacillus anthracis, Corynebacterium diphtheriae,Listeria monocytogenes, Clostridium tetani, or Clostridium difficile. 4.The method of claim 1, wherein the biofilm comprises Gram-negativebacteria.
 5. The method of claim 4, wherein the Gram-negative bacteriais one or more of Escherichia coli, Enterobacter species, Proteusmirablis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonellaspecies, Shigella species, Serratia species, Campylohacter species,Neisseria species, or Branhamella catarrhalis.
 6. The method of claim 1,wherein the surface is a tissue site.
 7. The method of claim 6, whereinthe tissue site is in contact with an indwelling medical device.
 8. Themethod of claim 1, wherein the surface is an indwelling medical device.9. The method of claim 8, wherein the indwelling medical device is acatheter or a ventilation tube.
 10. The method of claim 1, wherein therecombinant bifunctional ligand is formulated in a gel or a foam. 11.The method of claim 1, further comprising contacting the biofilm or thesurface with an antibiotic.
 12. The method of claim 1, furthercomprising contacting the biofilm or the surface with a protease. 13.The method of claim 1, wherein the biofilm and/or the surface is locatedin a wound.
 14. The method of claim 1, wherein thequorum-sensing-molecule-binding region may comprise at least one ofLuxR, ABC exporter, AgrC transmembrane sensor kinase, Lsr ABC-typetransporter, LuxQ transmembrane sensor kinase, ArpA, ComAB, and ComDtransmembrane sensor kinase.
 15. The method of claim 1, wherein thequorum-sensing-molecule-binding region and the protease-binding regionare chemically linked with a linker comprising a peptide of about 1 toabout 50 amino acids in length.